Our central goal is to understand the molecular basis for the chemical regulation of gap junction. Our central goal is to understand the molecular basis for the chemical regulation of gap junctions and its implications on cell behavior. This project has been a part of the Program Project Grant since its inception in 1990. For the next funding period, we will focus on four Specific Aims: 1) To begin a characterization of the mechanisms of gap junction formation and regulation in cells expressing Cx40 and Cx43. Cx40 and Cx43 do not form heterotypic channels. Yet, if both cells express both connexins, complex channels are formed. Moreover, some agonists regulate one isotype but not the other. We will determine whether a heteromeric connexin interacts with an opposing homomer and if so, whether the regulation of channel is dominated by one of the connexins. 2) To determine the pH sensitivity of gap junctions in cells expressing Cx40 and Cx43. We recently demonstrated that "hetero- domain interactions" between a truncated Cx40 and the heterologous CX43CT render the channel more pH sensitive than the wild-type Cx40. We therefore propose that in heteromeric Cx40-Cx43 gap junctions, hetero-domain interactions lead to synergism of pH grating. 3) To study the ability of connexin CT fragments to interfere with the chemical regulation of exogenous homomeric gap junctions. Previous studies show that injection of the entire Cx43CT fragment, or of a specific 17mer peptide to Cx43-expressing oocyte pairs can interfere with pH gating. Here, we will test whether these fragments can interfere with other regulatory functions of Cx43, or of Cx40. We will also seek to identify new peptides that interfere with Cx43 regulation. 4) To determine whether over-expression of the Cx43CT domain can lead to functional alterations in cell physiology and to the disruption of organ function. We have developed a transgenic mouse line that over-expressed the Cx43CT domain under the control of the cytomegaloviral (CMV) promoter (cardiac neural crest-targeted). We hypothesize that connexin fragments can disrupt tissue function without altering the expression of pore- forming proteins. Overall, we focus on how connexin-connexin interactions (between full-length molecules or between a connexin and a fragment) affect gap junction regulation. These studies will lead to a better understanding of the role of connexins in health and disease.